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. 2026 May 6;11(19):28128–28140. doi: 10.1021/acsomega.5c12731

Polysialic Acid Synthesis on the N- and O‑Glycans of Salmonid Polysialyltransferase ST8Sia IV

Anna Seidel †, Mathieu Decloquement ‡,§, Lin Sun ∥, Dorothée Vicogne ‡,§, Juliane Günther †, Christina E Galuska †, Somanath Kallolimath ∥, Sebastian P Galuska †,*, Anne Harduin-Lepers ‡,§,*
PMCID: PMC13191663  PMID: 42179534

Abstract

Extension of N- and O-glycans with linear sialic acid polymers (polySias) was described on a limited number of mammalian glycoproteins, including the α2,8-polysialyltransferases ST8Sia II and ST8Sia IV. Previous in vitro analyses have shown that ST8Sia IV from the salmonid fish Coregonus maraena (Cma) had high polysialylation activity and broader donor substrate specificity compared to its human counterpart. In this study, we investigated whether the fish ST8Sia IV was able to polysialylate N- and O-glycans of glycoproteins with a focus on its autopolysialylation capacity. Using a combination of strategies, including plant-based glycoengineering, we found that the Cma ST8Sia IV is able to use both types of acceptors for the formation of long polySia chains with a degree of polymerization of >40 consisting of N-acetyl­neura­minic acid and N-glycolyl­neura­minic acid. Given the importance of polySias in multiple health and disease states, the Cma ST8Sia IV represents a useful biocatalyst with applications in the fields of biosafe therapeutics and glycobiology.

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1. Introduction

The N- and O-glycans of glycoproteins play a central role in regulating numerous physiological processes in vertebrates, with the anionic sialic acid (Sia) residues of these carbohydrate structures playing a major role. Remarkably, Sia residues are present not only as monomers on glycoproteins but also as linear Sia polymers (polySias), which were first described in fish eggs. The elongation of N- and O-glycans with charged polySia chains is a special form of glycosylation taking place on a limited number of natural glycoproteins expressed in the central nervous system (CNS) and/or immune system. Polysialylated proteins have been described in several cell types, such as endothelial cells, smooth muscle cells, − fish eggs, and sperms, , as well as in body fluids including ejaculate, blood, and milk. Using murine knockout models of the responsible polysialyltransferases (polySTs), the significant biological impact of polySia was demonstrated in several physiological systems. For instance, polySia is essential for the formation of glomerular microvasculature and brain development. Based on its biological properties, several strategies have been developed for using polySia as a therapeutic agent. , For example, the molecule was suggested as a potential treatment for inflammation and neurodegeneration associated with Parkinson’s disease. Moreover, it was demonstrated that polysialylation of therapeutic proteins can increase their half-life.

The chemical structural diversity and complexity of polySia chains encountered in nature rely on their degree of polymerization (DP) and nature of Sias (i.e., N-acetylneuraminic acid (Neu5Ac), N-glycolylneuraminic acid (Neu5Gc), or 2-keto-3-deoxy-nonulo­sonic acid (KDN)) conferring both repulsive and adhesive properties to polysialylated proteins on the cell surface, as reviewed recently in ref . In mammal tissues, polySia is mainly a homopolymer of α2,8-linked Neu5Ac residues and has a highly variable DP. As an example, polySia chains consisting of more than 55 Sia residues were detected on the neural cell adhesion molecule NCAM in the postnatal mouse brain. PolySia chains identified in various fish species show remarkable species specificity associated with DP and Sia composition that could be related to the polysialylation machinery. , The elongation at position C8 of α2,3- or α2,6-linked Sia residues is catalyzed by an ancient family of α2,8-sialyltransferases (ST8SIA). Recently, a particular distribution of ST8SIA genes has been observed in teleost fish genomes. The human genome possesses two polyST genes ST8SIA2 and ST8SIA4, whereas salmonid fish possess three polyST genes st8sia2-r1, st8sia2-r2, and st8sia4, further suggesting a different α2,8-sialylation pattern of glycoproteins in fish tissues. Indeed, the ST8Sia IV cloned from the salmonid fish Coregonus maraena (Cma) shows high polysialylation activity, in contrast to both variants of Cma ST8Sia II. In addition, it has a broader donor substrate specificity using CMP-Neu5Ac, CMP-Neu5Gc, and CMP-KDN for the elongation of nascent polySia chains, compared to its human counterpart. , As mentioned above, polySia is of growing relevance for medical applications, , and the well-characterized Cma ST8Sia IV represents an attractive candidate for biotechnological exploitation. However, many other properties, including acceptor substrate specificity of fish polySTs, remain unclear. In contrast, mammalian polySTs have been well characterized, and their activity on a very limited number of N- and/or O-glycoproteins has been described. , As reviewed recently, natural glycoprotein acceptors for polysialylation on their N-glycans include primarily NCAM, the synaptic cell adhesion molecule 1 (Syn-CAM 1), a sodium ion channel, E-selectin ligand-1 (ESL-1), and quiescin sulfhydryl oxidase 2 (QSOX2). In contrast, O-glycan polysialylation in vivo is rare but documented: the glycoproteins neuropilin-2 (NRP-2) , and the polysialoglycoprotein (PSGP) in fish are exclusively polysialylated on O-glycans, and the chemokine receptor CCR7 is polysialylated on both N- and O-glycans. Remarkably, polySTs can also polysialylate their own N-glycans in vitro, and recently, polysialylated ST8Sia II and ST8Sia IV have been detected on human sperms and in serum, respectively. The autopolysialylation on N-glycans of mammalian ST8Sia II and ST8Sia IV, although not required, seems to positively influence their enzymatic activity on NCAM. Up to now, almost nothing has been known about such enzymatic properties of fish polySTs. In this study, we investigated, whether Cma ST8Sia IV is able to polysialylate N- and O-glycans with a focus on its autopolysialylation capacity. To this end, we used different protein expression strategies, including mammalian cell lines and a plant-based glycoengineering approach. The latter strategy is possible because plants were engineered using transient and stable expression methods to avoid plant-specific epitopes α1,3-fucose and β1,2-xylose, , resulting in complex post-translational modification similar to mammalian cells. , The plant-based approach was used to verify the results of the animal cell line and to test whether Cma ST8Sia IV can also be used in the area of “green biotechnology”. In combination with different analytical applications, we demonstrated that autopolysialylation of Cma ST8Sia IV occurs on both N- and O-glycans independently of the expression system, producing long polySia chains of comparable length to both Neu5Ac and Neu5Gc donor substrates.

2. Materials and Methods

2.1. Constructs

The Cma ST8Sia IV cDNA originally cloned in the p3 × FLAG expression vector was cloned into the pFUSE-rIgG-Fc2 vector (InvivoGen), a vector that expresses a fusion protein with a rabbit Fc-tag. To avoid polysialylation on the N-glycan of the Fc-tag, the Fc sequence was mutated at its N-glycosylation site. Asn75 was changed into Gln75 using a Q5 Site-Directed Mutagenesis Kit (New England Biolabs) with the primer pair 5′-GCAG­CAGTTC­cag­AGCAC­GATCC­GCGT­GGTC-3′ (sense) (mutation is written in lower case) and 5′-TCCC­GTAGC­GGCG­GCCGG-3′ (antisense), and mutagenesis was verified by sequencing at Microsynth Seqlab GmbH, Göttingen, Germany. To produce an active and soluble isoform of the enzyme, the Δ28ST8Sia IV sequence with its cytosolic and transmembrane domains deleted was amplified from the p3 × FLAG-ST8Sia IV plasmid, described in ref , using the primer pair 5′-gtcac­GAATTC­TGAACT­CGACAC­TTCAA­GACT­TATG-3′ (sense) and 5′-cttgct­AGATCT­cttgt­catc­gtcatc­AGATT­CGCAC­TTC­GAAG­TCG-3′ (antisense) (overhang and enterokinase cleavage site are written in lower case) and inserted into the mutated pFUSE-rIgG-Fc2 vector. The amplification and incorporation into the plasmid were verified by sequencing (Microsynth Seqlab GmbH, Göttingen, Germany).

To express a full-length isoform of the Cma ST8Sia IV in planta, the cDNA of the Cma ST8Sia IV was PCR amplified with primer pairs (Cma ST8Sia IV_F1: 5′-TATAT­CTAGA­ATGCG­TCTCT­CACGG-3′ and Cma ST8Sia IV_R1: 5′-TATAG­GATCCA­GATTCG­CACT­TCG-3′) carrying terminal restriction sites XbaI/BamHI. PCR products were cloned into the pPT2M binary vector carrying a C-terminal strep tag. The resulting plasmid pCma ST8Sia IV was transformed into Agrobacterium tumefaciens (strain GV3101 pMP90) and used for subsequent agroinfiltration experiments.

2.2. Cell Culture and Transient Transfection

HEK293 (ATCC CRL-1573) and N-acetyl­gluco­saminyl­trans­ferase I-negative HEK293S cells (ATCC catalog number CRL-3022) were obtained from LGC Standards (Molsheim, France). Cells were grown in DMEM (DMEM, Dutscher) supplemented with fetal calf serum (10% FCS, Biowest) at 37 °C and 5% CO2. Confluent cells (∼70%) were transiently transfected with 10 μg of p3 × FLAG-ST8Sia IV expression constructs in 100 mm Petri dishes with Lipofectamine 2000 (Thermo Scientific) in UltraMEM medium (Lonza) according to the manufacturer’s instructions. An empty p3 × FLAG-CMV9 was used as a control (Mock). Transiently transfected cells and cell culture media were collected 48 to 72 h after transfection. The cells were lysed in 100 μL of RIPA buffer (10 mM Tris-HCl; 150 mM NaCl; Triton X-100 1%, pH 6.4), 2 μL of 50× protease inhibitors by 10 aspirations and discharges with a syringe and a needle 26 G × 1/2 (Terumo). Recombinant enzymes produced in DMEM-FCS culture media were used without further purification as a crude enzyme source for enzymatic assays.

For the expression of Fc-tagged Cma ST8Sia IV, HEK293 and HEK293S cells were cultivated in DMEM or DMEM/Ham’s F12 medium (Capricorn Scientific GmbH, Ebsdorfergrund, Germany), respectively, both containing 10% IgG-depleted FCS (Capricorn Scientific GmbH, Ebsdorfergrund, Germany). The transfection was performed using the Xfect Transfection Reagent (Takara Bio Inc., San Jose, CA, USA) according to the manufacturer’s instructions. Cell culture supernatants were collected 72 h after transfection and stored at −80 °C.

2.3. Polysialylation Assays Using FLAG-ST8Sia IV

The donor substrates CMP-Neu5Ac and CMP-Neu5Gc were chemo-enzymatically synthesized as described previously , and verified using an LC-MS system equipped with a HILIC column as described for the analysis of milk oligosaccharides (Supplementary Figure S1). , Polysialylation assays were conducted for 2 h at 27 °C with 3 × FLAG-ST8Sia IV enzyme produced in either HEK293 cells, in 100 mM cacodylate buffer (10 mM MnCl2, 0.2% Triton-CF54) with 40 μM CMP-Sia (i.e., CMP-Neu5Ac or CMP-Neu5Gc). For further quantitative and analytical determination, polysialylation reaction was performed overnight at 27 °C in 1× cacodylate buffer (100 mM sodium cacodylate trihydrate, 10 mM MnCl2, 0.2% Triton-CF54) containing FLAG- and Fc-tagged ST8Sia IV using 40 μM CMP-Sia in a total volume of 250 μL.

2.4. SDS-PAGE and Western Blotting

Western blotting (WB) was used to visualize tagged recombinant Cma ST8Sia IV proteins secreted from transiently transfected HEK293 or HEK293S cells as previously reported. Briefly, culture media of transfected cells were boiled 5 min at 95 °C in 4× Laemmli buffer (235 mM Tris-HCl pH 6.8, 8% SDS, 40% glycerol, 10% β-mercaptoethanol, 0.01% bromophenol blue) and run on SDS-PAGE. After proteins were transferred onto Amersham Protran nitrocellulose membrane (GE Healthcare Life Sciences), the detection of recombinant ST8Sia IV proteins was achieved with the primary mouse anti-FLAG M2 antibody (1 μg/mL; Sigma-Aldrich). Images were acquired using a CCD camera (Fusion Solo, Vilber Lourmat) and Fusion software.

After the polysialylation reaction, polysialylated products treated or not treated by endoneuraminidase N (endoN) were heated for 5 min at 60 °C in 5× reducing sample buffer (Thermo Scientific). For Western blot (WB) analysis, proteins were separated using 7% SDS-PAGE under reducing conditions and subsequently transferred onto a PVDF membrane. To visualize polysialylated proteins, antibody staining was conducted, using 1 μg/mL anti-polySia mAb735 antibody (MHH, Hanover, Germany) as the primary and 0.13 μg/mL HRP-conjugated donkey anti-mouse (Jackson Immunoresearch) as the secondary antibody. To detect the rabbit Fc-tag, the membrane was stained with 19 ng/mL HRP-conjugated goat anti-rabbit IgG (Cell Signaling Technology). The chemiluminescence signal was detected using ECL Prime (Cytiva, Little Chalfont, UK) with a ChemDoc MP Imaging system (Bio-Rad, Feldkirchen, Germany).

For WB analyses of Cma ST8Sia IV expressed in planta, protein extraction and immunoblotting were performed as reported previously with modifications. In short, total protein (TP) was extracted in extraction buffer (100 mM Tris, 1 mM EDTA, 100 mM NaCl, and 40 mM ascorbic acid) containing 0.5% v/v Triton X-100. For endoN digestion, 50 μL of protein extracts (250 μg) was incubated with 0.5 μg of endoN on ice for 30 min. PNGase F digestion was carried out according to the manufacturer’s protocol (NEB, #P0704S). Approximately ∼50 μg (∼10 μL) TP samples, treated either with and without PNGase F or endoN, were heated for 10 min at 65 °C in 4× Laemmli buffer and resolved on 8% SDS-PAGE gel. Gels were used for immunoblotting. The blotted nitrocellulose membranes were incubated overnight at 4 °C with mAb735 diluted to 1:2000 in blocking buffer (3% nonfat milk in PBST, pH 7.4). After washing 3 times with 1× PBST, membranes were then incubated for 1 h with HRP-conjugated goat anti-mouse IgG secondary antibodies diluted to 1:10,000 in blocking buffer. Immunoblots were developed using Clarity Western enhanced chemiluminescence reagents (Bio-Rad Life Science).

2.5. Quantitative Analysis of 3 × FLAG-ST8Sia IV Polysialylation by HPLC Using CMP-Neu5Gc

The transfer of Neu5Gc residues onto Cma ST8Sia IV by polysialylation was quantified by an endoN approach using centrifugal filter units, as described previously. Briefly, 10 kDa ROTISpin MINI centrifugal filter units (Carl Roth GmbH + Co. KG, Karlsruhe, Germany) were prepared according to the manufacturer’s instructions. After polysialylation using CMP-Neu5Gc as substrate, samples were centrifuged at 14,000g at 4 °C until almost all the liquid was filtered and subsequently washed 5 more times with 250 μL of 50 mM NH4HCO3 to remove excess CMP-Neu5Gc. Thereafter, the volume was adjusted to 110 μL with 50 mM NH4HCO3 buffer, and endoN was added (1.34 μg/mL). In parallel, untreated samples of each CMP-Neu5Gc preparation were used as negative controls. For Western blotting, 10 μL of each sample was taken from the supernatant. The remaining sample was centrifuged, and the complete flow-through was dried in a vacuum concentrator and used for Sia quantification.

The Neu5Gc content of autopolysialylation samples was quantified using a reverse phase HPLC approach. The dried samples were hydrolyzed using 0.2 M trifluoroacetic acid (TFA) at 80 °C for 4 h. After drying in a vacuum concentrator, the samples were dissolved in 80 μL of DMB reaction buffer (2.7 mM DMB (Dojindo, Kumamoto, Japan), 9 mM sodium hydrosulfite, 0.5 M β-mercaptoethanol, and 20 mM trifluoroacetic acid) and incubated at 55 °C for 2 h. The reaction was stopped by adding 20 μL of 0.2 M NaOH.

The fluorescently labeled samples were analyzed using a Nexera HPLC system (Shimadzu) with a Superspher 100 RP-18 end-capped column (250 × 2 mm, Merck-Hitachi, Darmstadt, Germany). The eluents, 92% Milli-Q water, 4% acetonitrile, 4% methanol, and 0.1% TFA (E1) and 10% Milli-Q water, 45% acetonitrile, 45% methanol, and 0.1% TFA (E2), were run with a flow rate of 0.25 mL/min following the gradient t 0 min = 0% E2, t 2 min = 0% E2, t 25 min = 2% E2, t 35 min = 5% E2, t 40 min = 50% E2, t 45 min = 100% E2, t 50 min = 100% E2, t 51 min = 0% E2, and t 60 min = 0% E2. The fluorescence signals were detected using an extinction wavelength of 372 nm and an emission wavelength of 456 nm.

2.6. Affinity Precipitation and Chain Length Analysis

To determine the DP, a polysialylation reaction was performed with the FLAG-ST8Sia IV with 40 μM CMP-Neu5Ac or CMP-Neu5Gc in 100 mM cacodylate buffer and incubated at 27 °C overnight. After dialysis against Tris-buffered saline using Spectra/Por Dialysis Membrane Biotech CE Tubing (Spectrum Laboratories, MWCO: 50 kDa), the samples were precipitated using inactive endoN coupled to tosylactivated Dynabeads M-280 (Life Technologies, Oslo, Norway), according to the manufacturer’s instructions as described previously. After washing, the samples were eluted using 64.5 mM triethylamine and dried in a vacuum concentrator.

For chain length analysis, samples were derivatized using 40 μL of DMB reaction buffer overnight at 11 °C. The reaction was stopped by adding 10 μL of 1 M NaOH, and the samples were incubated for 1 h at room temperature to reverse lactonization. Subsequently, the fluorescence-labeled samples were injected into a Nexera HPLC system (Shimadzu, Duisburg, Germany) with a DNAPac PA-100 column (4 × 250 mm; 13 μm; Thermo Fisher Scientific, Waltham, MA, USA). Separation according to the DP was performed using Milli-Q water (E1) and 2 M ammonium acetate (E2) with a flow rate of 1 mL/min, following the gradient t 0 min = 0% E2, t 5 min = 0% E2, t 15 min = 8% E2, t 20 min = 11% E2, t 35 min = 16% E2, t 55 min = 23% E2, t 95 min = 31% E2, t 130 min = 40% E2, t 131 min = 100% E2, t 140 min = 100% E2, t 141 min = 0% E2, and t 175 min = 0% E2. Fluorescence signals were detected by using an extinction wavelength of 372 nm and an emission wavelength of 456 nm.

2.7. Autopolysialylation of Fc-tagged Cma ST8Sia IV on Protein A Magnetic Beads

Magnetic Protein A Dynabeads (Invitrogen) were washed three times with PBS and incubated for 1.5 h with double the volume of the Fc-ST8Sia IV cell culture supernatant at room temperature on a rotator. Thereafter, the beads were washed again three times with PBS and once with cacodylate buffer. The polysialylation reaction was performed in cacodylate buffer with 40 μM CMP-Neu5Ac or CMP-Neu5Gc rotating overnight at 27 °C. The beads were washed three times with 50 mM NH4HCO3 to remove excess CMP-Sia, and the Fc-ST8Sia IV was finally eluted for 10 min using 64.5 mM triethylamine and subsequently dried in a vacuum concentrator. The samples were analyzed by Western blotting, as described above.

2.8. Agroinfiltration In Planta of Cma ST8Sia IV and Enrichment of Polysialylated Proteins by Ion Exchange (IEX) Resin

The Nicotiana benthamiana (N. benthamiana) plant was engineered by introducing the mammalian sialylation pathway or mucin-type O-glycan pathways and downregulating respective glycosyltransferase using RNAi and also by CRISPR-Cas9, as previously described. − , The glycosylation mutant plants (ΔXTFT) were used to produce recombinant proteins with mammalian-like glycans. Agrobacterium carrying Cma ST8Sia IV and genes involved in the two mammalian N-Sia and mucin-type O-Sia pathways, N-Sia (GNE: N-acetylglucosamine-epimerase, NANS: N-acetyl neuraminic acid synthase, CMAS: CMP-Sia synthase, SLC35A1: CMP-Sia transporter, B4GALT1: β-1,4-galactosyltransferase 1, and ST6GAL1: α-2,6-sialyltransferase) and O-Sia (genes involved in CMP-Sia synthesis and transporter (GNE, NANS, CMAS, and SLC35A1) and ppGALNT2: peptidyl GalNAc transferase 2, C1GALT1: β-1,3-galactosyltransferase, ST3GAL1 (α-2,3-sialyltransferase), and ST6GALNAC4 (α-2,6-sialyltransferase)), were coexpressed to achieve polysialylation on N-glycans and O-glycans, respectively, as previously described. To prepare the agroinfiltration mixture, Agrobacterium carrying the genes of interest, i.e., Cma ST8Sia IV and N- and mucin-type O-glycan Sia pathway constructs, were grown at 29 °C overnight. Agrobacteria cultures were resuspended in infiltration buffer (10 mM Mg2SO4, 10 mM MES; pH 5.6) with an optical density (OD600) of 0.1 for pCma ST8Sia IV and 0.05 for N- and O-sialylation pathway enzymes. The agroinfiltration mix was delivered into 4- to 5-week-old N. benthamiana plants using a needleless syringe.

To enrich polysialylated protein fractions, total protein (TP) was extracted from plant leaves expressing Cma ST8Sia IV with either N- or mucin-type O-sialylation pathway constructs. Approximately 20 mL of TP containing polysialylated protein was applied to enrich using ion exchange (IEX) resin (Biorad). The manually packed column was pre-equilibrated with 10 column volumes (CV) of 1× PBS (137 mM NaCl, 3 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4; pH 7.4) at a flow rate of 1.5 mL/min. Washing was done with 20 CV of 1× PBS (pH 7.4). The bound polysialylated proteins were eluted in elution buffer (1 M NaCl in PBS; pH 7.4). To check the efficiency of the enrichment method, the intermediate fractions were monitored by WB (Supplementary Figure S2). In addition, the chain length was determined as described above (section ).

2.9. Statistical Analysis

Statistical analysis was performed using R Statistical Software (v.4.3.1). Two-way ANOVA with Tukey’s multiple comparisons test was performed using the package MultcompView (v.0.1.10). Asterisks indicate the p-value: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001; n.s. means not significant. Plots were created using the Tidyverse package (v.2.0.0) and Patchwork package (v.1.3.0.9000).

3. Results and Discussion

3.1. Cma ST8Sia IV Polysialylates N- and O-Glycans

Human ST8Sia IV is able to polysialylate N- and O-glycans of proteins. , Data obtained in a previous study suggested that the fish polyST Cma ST8Sia IV is also able to transfer Sia to glycoproteins decorated with N- and/or O-glycans using the homemade unnatural sugar donor CMP-SiaNAl (cytidine-5′-mono­phospho-N-4-penty­noyl­neura­minic acid) and a microplate sialyltransferase assay (MPSA). However, only a general addition of Sia could be determined with the applied MPSA , by the increase of the fluorescence signal, and it was not possible to distinguish whether only one Sia residue was transferred or whether a longer polySia chain could be synthesized. In addition, similarly to the human ST8Sia IV, the Cma ST8Sia IV possesses 5 potential N-glycosylation sites (Figure A) on evolutionary conserved sequon (N-X-S/T) positions (Supplementary Figure S3), but it is not known whether complex N-glycans on Cma ST8Sia IV are necessary for enzymatic activity. To tackle these issues, we produced Cma ST8Sia IV in two distinct HEK293 cell lines. In addition to regular HEK293 cells equipped with the enzymatic machinery to synthesize sialylated N- and O-glycans, we used HEK293S cells. These HEK293S cells are defective in the N-acetyl­gluco­saminyl­trans­ferase 1 gene MGAT1, abrogating the formation of complex-type N-glycans (Figure B). In HEK293S cells, (poly)­sialylation of recombinant N-glycoproteins is not possible, and it can take place only on O-glycoproteins.

1.

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Cma ST8Sia IV polysialylates N- and O-glycoproteins. (A) Schematic representing the protein structure of Cma ST8Sia IV with localization and amino acid sequence of all 5 N-glycosylation sites predicted at positions Asn 50, 73, 118, 203, and 218. Positions of the transmembrane domain (TM) and sialylmotifs L, S, III, and VS are indicated. (B) Schematic representation of complex-type N-glycans of glycoproteins in HEK293 cells (upper) and oligomannose-type N-glycans of glycoproteins in MGAT1-deficient HEK293S cells (lower). Monosaccharides are represented according to the SNFG representation of glycans. (C) The Cma ST8Sia IV produced as a truncated form in the supernatant of transfected HEK293 and HEK293S cells were subjected to PNGase F treatment for various time points ranging from 0 to 30 min. An empty vector (Mock) was used as a negative control in both cell lines. After 5 min at 95 °C, proteins were separated on 8% SDS-PAGE and transferred onto a nitrocellulose membrane. WB was carried out with the anti-3 × FLAG antibody. Arrows on the right side represent the various N-glycosylation sites detected. The complete areas of the WB are shown in Supplementary Figure S3, including 5 additional time points (45, 60, 90, 120, and 240 min). (D) The initial polysialylation status of Cma ST8Sia IV produced in HEK293 or HEK293S cells was visualized by WB (left WB). Aliquots of the samples were treated with PNGase F to remove N-glycans. After 5 min at 65 °C, proteins were separated on 8% SDS-PAGE and transferred onto a nitrocellulose membrane. WB was carried out using the mAb735. The polysialylation status of glycoproteins after the sialylation reaction using 40 μM CMP-Neu5Ac was analyzed by WB in the same way (right WB). Molecular weight markers are indicated on the left side.

We checked that MGAT1 deficiency in HEK293S cells did not influence the secretion level of the recombinant Cma ST8Sia IV compared to HEK293 cells. The recombinant N-terminally FLAG-tagged enzyme produced in the supernatant of both cell lines could be detected by WB using an anti-FLAG antibody at ∼55 kDa (Figure B). Interestingly, the anti-FLAG signal obtained for the enzyme produced in HEK293 cells (upper) appears to be slightly fuzzier than the one obtained for the enzyme produced in HEK293S cells (lower), suggesting higher heterogeneity of glycosylation of this recombinant enzyme. The release of N-glycans with PNGase F, which cleaves between the innermost GlcNAc and asparagine residues of high mannose, hybrid, and complex-type N-glycans, resulted in a shift to ∼40 kDa, confirming the post-translational modification of the enzyme with N-glycans in both cell lines. The time course analysis of the enzymatic N-glycan release further suggested that predicted N-glycosylation sites were occupied by N-glycans in both cell lines, since different bands become detectable in a time-dependent manner (Figure C, Supplementary Figure S3). However, the number of occupied glycosylation sites remains ambiguous because differently glycosylated enzymes may exhibit identical migration patterns.

We then investigated the polysialylation status of the recombinant enzymes produced in the supernatant of both cell lines using the mAb735 antibody. Interestingly, a faint polySia signal could be detected for both culture media, indicating a small proportion of initial polysialylation (Figure D, left), which was not observed in the Mock-transfected culture supernatant. These signals did not disappear but were slightly shifted to lower molecular weight after PNGase F treatment, further indicating the production of active and polysialylated Cma ST8Sia IV enzymes in both cell lines (Figure D, left).

It has long been known that the HEK293 culture supernatants contain sialylated glycoproteins from the DMEM-FCS culture media as well as additional physiologically secreted biomolecules such as the well-known polySia carrier NCAM. It was therefore desirable to check if polysialylation could take place in the absence of sialylated N-glycans on ST8Sia IV from HEK293S cells. For that purpose, we performed in vitro 2 h polysialylation reactions using the recombinant enzymes secreted from both cell lines and 40 μM CMP-Neu5Ac. This led to higher polySia signals detected with the sensitive anti-polySia mAb, although they were less smeary and intense for the enzyme produced in HEK293S cells (Figure D, right). Interestingly, even if a large polySia signal disappeared after PNGase F treatment, indicating the release of polysialylated N-glycans, lower levels of polysialylation were still detectable (Figure D, right). This remaining polySia signal could indicate an incomplete release of the N-glycans or, more likely, that O-glycoproteins are also polysialylated. Remarkably, when ST8Sia IV from HEK293S was tested, the polySia signal intensity even slightly increased after N-glycan release (Figure D, right). This might be explained either by a better accessibility of the mAb735 to the polySia O-glycans or by a more focused migration of the polySia carrier after release of the N-glycans by PNGase F. Even if the signal increase cannot be clarified, the observed polysialylation signal demonstrates that despite the absence of complex N-glycans, the recombinant enzyme conserves enzymatic activity and remains able to polysialylate glycoproteins. Moreover, the PNGase F experiment strongly suggests that O-glycoproteins were the only acceptors for the ST8Sia IV from the HEK293S culture supernatant. Collectively, these data show that complex N-glycans on Cma ST8Sia IV are not necessary for the qualitative enzymatic activity of the polyST and that the enzyme is able to polysialylate both N- and O-glycans on glycoproteins, even though no conclusion can be drawn relative to the quantitative enzyme activity.

3.2. Cma ST8Sia IV Catalyzes the Transfer of Neu5Gc Residues on N- and O-Glycans

We have shown previously that Cma ST8Sia IV, when produced in HEK293 cells, can utilize CMP-Neu5Gc in addition to CMP-Neu5Ac for polysialylation. To test whether Cma ST8Sia IV produced in HEK293S can also incorporate Neu5Gc into O-glycans of glycoproteins, we performed polysialylation reactions in centrifugal filter units with or without CMP-Neu5Gc. After the remaining CMP-Neu5Gc was washed away, aliquots of the reaction products were analyzed by Western blotting to visualize polySia. As shown in Figure A, we observed that the Cma ST8Sia IV produced in HEK293S cells could also use CMP-Neu5Gc for polysialylation of O-glycans of glycoproteins, as previously shown for the Cma ST8Sia IV produced in HEK293 cells. To verify the incorporation of Neu5Gc, the remaining samples of the polySia reaction products in the basket of the centrifugal filter units were degraded by endoN. Following centrifugation, the polySia degradation products in the flow-through were hydrolyzed and fluorescently labeled with DMB for HPLC analysis. Our data showed a significant increase of Neu5Gc upon incubation with CMP-Neu5Gc (Figure B), whereas no significant increase of the Neu5Gc values was observed with no addition of CMP-Neu5Gc. Using an antibody-independent HPLC approach, we showed that Cma ST8Sia IV could efficiently use CMP-Neu5Gc as a donor substrate for polysialylation, regardless of the cell line in which the enzyme was produced and the target for the polysialylation of N- or O-glycoproteins. Human cells do not synthesize Neu5Gc de novo due to a deletion acquired during human evolution in the cytidine mono­phospho-N-acetyl­neura­minic acid hydroxylase (CMAH) gene, and an unknown number of monoSTs are still able to transfer Neu5Gc to nascent glycoconjugates. This had several consequences in the human sialobiology including a loss of recognition ability of NeuGc by human siglecs, a lost ability of the human ST8Sia IV to transfer various Sias, , and the formation of anti-Neu5Gc antibodies. The combination of anti-Neu5Gc antibodies, the uptake of Neu5Gc by diet, and its incorporation into glycoconjugates are discussed to accelerate inflammation or even cancer in humans. , This would severely limit their use in humans for medical purposes, for example, in intravenous applications. However, an external therapeutic application of Neu5Gc-containing polySia would be possible. One possibility would be its use in the manufacture of antimicrobial plasters or gels since numerous antimicrobial peptides bind to the hydrophilic and negatively charged polySias without a loss of their antimicrobial activity. −

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Neu5Gc is incorporated into polySia chains by Cma ST8Sia IV produced in HEK293 and HEK293S cells. Polysialylation reactions were performed in a filter unit overnight with or without 40 μM CMP-Neu5Gc. Polysialylated proteins were either (A) separated on SDS-PAGE, and WB was obtained with the mAb735 showing polysialylation obtained with ST8Sia IV produced in HEK293 (upper) and HEK293S cells (lower), or (B) treated (red boxplots) with endoN, collected in the flow-throughs, and analyzed by HPLC. As an additional control setup, the flow-throughs of samples without endoN were analyzed (blue boxplots). Samples from 3 independent experiments were measured (n = 3). The asterisks indicate the p-value: *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001; n.s. stands for not significant.

3.3. Cma ST8Sia IV Generates PolySia Chains of DP > 40 on Its N- and O-Glycans

Moreover, the chain lengths of the enzymatic products were analyzed by an additional HPLC approach. To this end, the polysialylated molecules were enriched with enzymatically inactive endoN coupled to magnetic beads. Such an affinity precipitation is possible since endoN contains a binding domain for oligo and polySia chains. , The polySia chains of the precipitated glycoproteins were released, fluorescently labeled, and separated according to their DP by anion exchange chromatography. The detected polySia chains of Cma ST8Sia IV of both cell lines, using CMP-Neu5Ac as a donor substrate, reached chain lengths of more than 40 Sia residues (Figure A and Supplementary Figure S4A). However, even the pre-existing polySia chains (samples without CMP-Neu5Ac added as a substrate) showed comparable chain lengths. Nevertheless, the quantity of all of the detected chain lengths increased with the addition of CMP-Neu5Ac. The results are in line with the WB shown in Figure D. However, to ensure valid comparisons, enzymatic reactions must be performed in parallel using the same enzyme preparation, with and without CMP-Neu5Ac. Consequently, the 0 μM control values from different supernatants are not directly comparable. Remarkably, when CMP-Neu5Gc was used as a donor substrate, additional peaks appeared in chromatograms (Figure B and Supplementary Figure S4B). Thus, in addition to the initially present Neu5Ac homopolymer, Neu5Gc-containing chains with a changed retention time were detected. It was already described that the retention time of a polySia chain depends on its composition. , To improve the visualization of the Neu5Gc-containing peaks, we subtracted the chromatograms of the control samples (initial polyNeu5Ac signals; Supplementary Figure S5). Interestingly, we observed negative peaks at the retention time corresponding to oligo/polyNeu5Ac peaks likely because these initial chains were used as substrates for further extension. As these initial chains are elongated, their original peaks decrease, resulting in a reduced or negative peak area. This suggests that oligo/polyNeu5Ac chains were elongated with Neu5Gc and that a certain proportion of Neu5Gc must be present in order to influence the retention time. However, analysis of the chromatograms does not enable us to determine whether it was only oligo/polyNeu5Ac chains of glycans that were elongated with Neu5Gc, or whether Neu5Ac monomers of glycans were also used as acceptors. This would be also difficult to assess with mass spectrometry-based techniques since the chains must be first released for the sensitive detection of polySia chains, and an internal degradation can occur. , Therefore, this remains an open question due to limited polySia analytic approaches and highlights the need for the development of more MS-based analyses. To summarize, regardless of the cell line in which Cma ST8Sia IV was produced, CMP-Neu5Gc can be used to form Neu5Gc-containing polySia with a DP > 40. Since only O-glycans are polysialylated with the enzyme source from HEK293S cells (Figure B), Cma ST8Sia IV seems to generate polySias with comparable chain lengths on both N- or O-glycans. Heteropolymers of Neu5Ac and Neu5Gc with a DP ranging from 2 to 20 were observed on the PSGP of salmonid fish eggs. The differences in chain length could be explained either by a higher polymerase activity of the Cma enzyme, different conditions between in vitro and in vivo polysialylation, or the use of a more efficient analytical method for the detection of polySia chains. The obtained length of polySia chains generated by the fish enzyme in in vitro polysialylation reaction is quite important (DP > 40). These data underscore both the robustness of this fish enzyme and its relevance for glycoengineering for medical applications since the interaction between polySia and several bioactive molecules depends on the chain length. For instance, lactoferrin and histones need chains consisting of more than 20 sialic acid residues for efficient binding. ,,, Although the mechanism of polySia chain termination remains unknown, it has been proposed to rely on the interaction of the PSTD domain of the mammalian ST8Sia II and ST8Sia IV with the extended polySia chains of NCAM; , there would be a loss of affinity of the enzyme for its protein substrate as DP increases. In vivo, the concentration of CMP-Sia donors (CMP-Neu5Ac or CMP-Neu5Gc) could be an activator (or an inhibitor) depending on the affinity of the enzyme. In addition, heterodimers of ST8Sia II and ST8Sia IV are believed to exist during embryogenesis that would lead to an increase of polySia chain length on NCAM, although polySTs can work independently.

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Cma ST8Sia IV produced in HEK293 and HEK293S cells can use CMP-Neu5Ac and CMP-Neu5Gc to elongate polySia chains. The precipitates of polysialylation reactions conducted with Cma ST8Sia IV produced in HEK293 cells or HEK293S cells with (A) 40 μM CMP-Neu5Ac (purple line) or (B) 40 μM CMP-Neu5Gc (blue line) were used for “mild” DMB labeling and analysis by anion exchange chromatography. In addition, a polysialylation reaction without an added donor substrate (black line) was performed. The chain lengths are given for selected peaks. Magnifications for visualizing chains in the DP 40 range are shown in Supplementary Figure S4.

3.4. Cma ST8Sia IV Polysialylates Its N- and O-Glycans

Using cell culture supernatants, it is difficult to distinguish Cma ST8Sia IV autopolysialylation from other glycoprotein polysialylations. Since FLAG-tagged ST8Sia IV cannot be purified without a significant loss of its activity, we adopted another strategy using an ST8Sia IV Fc-fusion protein. This strategy was described by Mühlenhoff and colleagues to characterize the autopolysialylation of mammalian ST8Sia IV (Figure A). The Fc-tagged enzyme was efficiently purified with Protein A-containing magnetic beads, as proven by Coomassie-stained SDS gel, where the purified ST8Sia IV from both cell lines showed a distinct band at ∼70 kDa (Figure B). Purified ST8Sia IV from both cell lines was used to assay its autopolysialylation capacity overnight in centrifugal filter units with or without CMP-Neu5Ac. Reaction products were run on SDS-PAGE and visualized by WB using the mAb735. As shown in Figure C, a small amount of polysialylated ST8Sia IV from both sources was detectable with no CMP-Neu5Ac added, and increased staining intensity was observed upon CMP-Neu5Ac addition, demonstrating that the isolated Fc-fusion protein is active on glycans of ST8Sia IV independent of the cell line. The release of the N-glycans by PNGase F led to a decrease of the polySia signal in the case of ST8Sia IV produced in HEK293 cells (Figure D), demonstrating that N-glycans, when present, are prevalently polysialylated, whereas a slight increase of the polySia signal was observed in the case of ST8Sia IV produced in HEK293S cells as previously observed (Figure D, right).

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Cma ST8Sia IV autopolysialylates its own N- and O-glycans. (A) Workflow illustrating the purification steps for Fc-tagged ST8Sia IV. Created with BioRender.com. (B) The recombinant Cma Fc-tagged ST8Sia IV produced in the supernatant of HEK293 and HEK293S cells was isolated with Protein A magnetic beads and visualized by Coomassie staining after SDS-PAGE. (C) Isolated Cma Fc-tagged ST8Sia IV was used for autopolysialylation reactions overnight with or without CMP-Neu5Ac. Reaction products were run on SDS-PAGE and visualized by WB using the anti-polySia mAb735. EndoN-treated samples were used as a negative control. (D) In addition, polysialylated products were treated by PNGase F and/or endoN, run on SDS-PAGE, and visualized by WB with mAb735 against polySia or (E) with an HRP-conjugated goat anti-rabbit IgG antibody against the Fc-tag of Cma ST8Sia IV.

In addition to immunostaining against polySia, the same sample setup was employed to target the protein part of Cma ST8Sia IV. For this purpose, an HRP-conjugated goat anti-rabbit IgG targeting the Fc-tag was used for Western blotting. In line with the Coomassie staining, a strong signal was observed for unpolysialylated Fc-tagged Cma ST8Sia IV but not for the polysialylated version (Figure E). The absence of a signal for polysialylated ST8Sia IV might be due to the fact that polysialylated proteins are often more difficult to detect than their unpolysialylated counterparts. It has been suggested that polySia chains may inhibit antibody binding to the protein backbone. This hypothesis is confirmed by comparing the bands of untreated samples with those treated with endoN. After the degradation of polySia, the protein bands thickened only at the top. Thus, an increase in the amount of Fc-tagged Cma ST8Sia IV with a higher molecular weight was observed after endoN treatment. This effect has been described previously and is due to Sia oligomers remaining on the protein after polySia degradation. However, the differences between the untreated and endoN-treated samples of HEK293 cells are less pronounced, when the N-glycans are released simultaneously by PNGase F (Figure E, lanes 3 and 4), since this Cma ST8Sia IV contains polysialylated N- and O-glycans (Figure D, lanes 1 and 3). In general, the results show that only a small proportion of Cma ST8Sia IV undergoes autopolysialylation.

Collectively, these results suggest that in the presence of sialylated N-glycans, a small ratio of O-glycans is autopolysialylated, whereas O-glycans are likely more accessible and therefore strongly autopolysialylated in the absence of sialylated N-glycans.

3.5. Structural Analysis of N- and O-Glycans of Cma ST8Sia IV Produced in Plants

Remarkably, it was described that salmonid ST8Sia IV of rainbow trout (Oncorhynchus mykiss) exhibits no autopolysialylation activity in vitro; and no autopolysialylation of the fish polySTs was reported in vivo. This could be due to structural differences between the two salmonid variants of ST8Sia IV or to the use of different analytical strategies. To verify the autopolysialylation activity of Cma ST8Sia IV, the autocatalytic activity was examined in a plant expression system. To this end, the enzyme was coexpressed with two distinct mammalian sialylation pathways (N- and O-Sia pathways) in N. benthamiana leaves. ,

Total protein extracts were monitored at 3 days post infection (dpi) by WB using the anti-polySia mAb. No polySia-specific signal was observed when Cma ST8Sia IV was expressed alone, while upon coexpression with the N- or O-Sia pathway, Cma ST8Sia IV exhibited polySia-specific signals (Figure A). To confirm, whether the polySia signal was associated with N- or O-linked glycans, samples were treated with PNGase F. Following PNGase F treatment, the polySia signal disappeared in the Cma ST8Sia IV sample coexpressed with the N-Sia pathway but remained unchanged in the sample coexpressed with the O-Sia pathway. Treatment with the polySia-specific neuraminidase endoN abolished the smear signals in samples coexpressing either the N- or O-Sia pathway, confirming the presence of polySia. In line with the experiments with the Fc-tagged ST8Sia IV containing complex or oligomannose-type N-glycans (Figure ), these findings indicate that, in addition to N-glycans, Cma ST8Sia IV also contains O-glycosylation sites that are subject to autopolysialylation.

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Cma ST8Sia IV expression in a heterologous plant system. (A) The total protein (TP) extracts from leaves infiltrated with Cma ST8Sia IV constructs with two distinct mammalian Sia pathways, N-Sia (upper) and O-Sia pathway (lower), were monitored by WB using mAb735. In addition, aliquots of PNGase F- or endoN-treated samples were analyzed. TP from leaves infiltrated without Cma ST8Sia IV was used as a negative control. (B) Polysialylated proteins extracted from leaves were enriched by ion exchange resin and inactive endoN affinity precipitation. Chain length analysis by anion exchange HPLC was performed for Cma ST8Sia IV with the N-Sia pathway and Cma ST8Sia IV with the O-Sia pathway.

To analyze the autopolysialylation of N- and O-glycans in more detail, the DP of polySia was determined. Therefore, the polySia chains on Cma ST8Sia IV were analyzed by HPLC as described above. The chain length analysis revealed polySia chains with more than 40 Sia residues on the N- and O-glycans (Figure B). Thus, independent of the acceptor glycan of Cma ST8Sia IV, an efficient elongation of the polySia chains occurred in the applied plant system. Autopolysialylation on N-glycans was observed in vivo only for mammalian ST8Sia IV. While the autopolysialylation of Cma ST8Sia IV on its N- and O-glycans is biochemically feasible, its occurrence in vivo remains uncertain. Even if polySia was detected in several fish tissues, ,,, the observed O-glycan autopolysialylation of recombinant Cma ST8Sia IV likely reflects a combination of the intrinsic catalytic activity of the enzyme, high local donor availability, and sustained expression levels in a recombinant system.

4. Conclusions

We studied here the complexity and uniqueness of salmonid fish polysialylation machinery. We have shown that Cma ST8Sia IV is able to polysialylate N- and O-glycans of glycoproteins. In contrast to its human orthologue, the fish enzyme is able to use either CMP-Neu5Ac or CMP-Neu5Gc to generate polySia chains. We focused on its autopolysialylation capacity and used different strategies, including a plant-based glycoengineering approach, to demonstrate that the Cma ST8Sia IV is able to use either its own N- or O-glycans as acceptors. Our analytical study showed that Cma ST8Sia IV catalyzes the formation of long chains of polySia polymers with a DP > 40. Whether these novel modifications on the fish ST8Sia IV have functional consequences remains unknown. However, the results demonstrate that Cma ST8Sia IV is a promising biotechnological tool to synthesize different types of polySias with potential for various biomedical applications.

Supplementary Material

ao5c12731_si_001.pdf (709.8KB, pdf)

Acknowledgments

We thank Martina Mühlenhoff (Medizinische Hochschule Hannover, Institut für Klinische Biochemie, Hannover, Germany) for the kind gifts of inactive and active endoN, as well as mAb735. The contribution of the COST Action CA18103-INNOGLY supported by the European Cooperation in Science and Technology (COST) is greatly acknowledged. The Abstract graphic was created with BioRender.com.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c12731.

  • HILIC-MS analysis of CMP-Neu5Ac and CMP-Neu5Gc; WB analysis of intermittent fractions of ion exchange enriched polySia; C. maraena ST8Sia IV N-glycosylation sites; zoomed-in chromatograms of Figure ; and chain length analysis chromatograms with background compensation (PDF)

∇.

A.S. and M.D. contributed equally to this work. A.S., M.D., D.V., J.G., C.E.G., and L.S.: Performed experiment, data curation, data analysis, and writing original manuscript draft. A.H.-L., S.P.G., and S.K.: Designed experiment, data analysis, funding acquisition, supervision, and writing original and revision manuscript.

This work was supported by the CNRS and the University of Lille to A.H.-L., the ANR-21-CE44-0032 (project PsaMar), the Deutsche Forschungsgemeinschaft (GA 1755 5–1) to S.P.G., and the PHC Procope grant (project no. 42533RC) to A.H.-L. and S.P.G. Moreover, this work was supported by grants of the Austrian Science Fund appointed to S.K. (P37211/grant DOI: 10.55776/P37211 and TAI7170424/grant DOI: 10.55776/TAI7170424).

The authors declare no competing financial interest.

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